Charles University in Prague Faculty of pharmacy in Hradec Králové Department of Biological and Medical science Candidate: Magdaléna And lová Supervisor: Mgr. Marcela Vejsová, Ph.D. Name of diploma thesis: Evaluation of activity of potentional antibacterial substances throught of the use microdilution broth metod Background Aim of this diploma work was research of activity of potentional antimicrobiotic substances. Research of antibacterial substances is one of the most important factor in pharmaceutic industry. The main reason is being the never stopping growth of bacterial resistence. Methods The microdilution broth metod was used to test the substances. This metod was used because of low difficulty and low cost. All the steps including the final analising was done by hand. Results The substances were divided in groups depending their chemical struction. The most effective was the salicylanilide derivates group. The other groups inhibitated growth of bacteria very little or were non-functional. In case of every bacterial stems were analised all substances which were effective. Conclusion Depending on the results the most sensitive and the most resistant bacterial stem were choosen. The most sensitive reaction on tested substances had bacterial stem Staphylococcus aureus. The Klebsiella pneumoniae...

Multiresistens hos gramnegativa bakterier är ett stort problem i stora delar av världen. Avsaknaden av nya preparat har resulterat i återintroduktion av ett tidigare stoppat antibiotikum, kolistin, men dessvärre kantas resistensbestämningen av flertalet problem. Kolistin är en positivt laddad molekyl som binder till lipid A i yttermembranet, vilket destabiliseras och resulterar i celldöd. Flertalet resistensmekanismer mot kolistin finns beskrivna och majoriteten resulterar i förändrad lipid A-struktur, vilket leder till försämrad kolistininbindning. Syftet med examensarbetet var att utvärdera fyra potentiella screeningmetoder för att upptäcka kolistinresistens och jämföra resultaten mot buljongspädning som referensmetod. De fyra screeningmetoderna var buljongeluering och diskdiffusion samt de kommersiellt tillgängliga screeningtesterna Superpolymyxin och Rapid polymyxin NP. Totalt analyserades 57 helgenomsekvenserade Enterobacterales, varav 28 Escherichia coli och 29 Klebsiella pneumoniae, där aktuella resistensmekanismer var kända. Samtliga isolat analyserades en gång per metod och utöver detta analyserades även tre kontrollstammar. Vid buljongspädning erhölls värde för minsta inhiberande koncentration (MIC) kolistin, vilket användes för klassificering som känslig (MIC ≤ 2 mg/l) eller resistent (MIC > 2 mg/l). De fyra screeningtesterna var designade för att upptäcka resistenta isolat och innehöll 2–4 mg/l kolistin. Resultaten från screeningtesterna var kvalitativa och känslighetskategoriserade isolaten. Sensitivitet och specificitet beräknades för metoderna. Högst sensitivitet (100 %) noterades för Superpolymyxin och Rapid polymyxin NP, och lägst specificitet (76 %) för buljongeluering. Med hänsyn till resultat, prevalens, hållbarhets-, tids- och kostnadseffektivitet drogs slutsatsen att Rapid polymyxin NP är bästa alternativet som screeningmetod av utvärderade metoder. / Infections due to multidrug resistant Gram-negative bacteria are a major problem in large parts of the world. Due to lack of treatment options, a previously banned antibiotic, colistin, has been reintroduced. Unfortunately, susceptibility testing of colistin is problematic and currently the only recommended method is broth microdilution (BMD). Colistin is a positively charged molecule that binds to lipid A at the negatively charged outer cell membrane, which leads to cell disruption. Several mutations causing colistin resistance have been identified, most of them cause alterations of lipid A, resulting in impaired colistin outer cell membrane interaction. The aim of this study was to evaluate four potential screening methods for colistin resistance and compare the results with BMD (reference method). The four screening methods were broth disk elution, disk diffusion and the commercially available tests Superpolymyxin and Rapid polymyxin NP. A collection of 57 whole genome sequenced Enterobacterales consisting of 28 Escherichia coli and 29 Klebsiella pneumoniae were included in the study. Reference results were obtained by BMD, where the estimated minimum inhibitory concentration (MIC) values were used for classification of the isolates as sensitive (MIC ≤ 2 mg/l) or resistant (MIC > 2 mg/l). Sensitivity and specificity were calculated for the methods. The highest sensitivity (100 %) was noted for Superpolymyxin and Rapid polymyxin NP, and the lowest specificity (76 %) for broth disk elution. Considering the results, prevalence, durability, time and cost efficiency, it was concluded that Rapid polymyxin NP is the best alternative as a screening method of evaluated methods.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of thesis: Evalution of activity of potentional antimycotic substances through the use of microdilution broth method III Autor of thesis: Alena Zavřelová Supervisor: Mgr. Klára Konečná, Ph.D. Background: The aim of this thesis, due to the growing incidence of fungal diseases and the growing incidence of resistance, was to determine the activity of potentially antimycotic active substances. We tested newly synthesized compoused, structurally based on the substance of pyrazinamide which is significant antituberculotic. Methods: Testing of substances was carried out using a broth microdilution method on eight strains of pathogenic fungi consisting of five yeast strains: Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Trichosporon asahii and three strains of filamentous fungi: Aspergillus fumigatus, Absidia corymbifera, Trichophyton mentagrophytes. Results: From the range of tested concentrations and from the total number of the thirty tested substances, the most noticeable antifungal aktivity was revealed in the substance of 5-methyl-6-(2-phenylhydrazinyl)pyrazine-2,3- dicarbonitrile. All strains, except Trichophyton mentagrophytes strain, showed a relatively...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biological and medical sciences Study program: Pharmacy Autor: Klára Kopečná Supervisor: Mgr. Klára Konečná, Ph.D. Title of diploma thesis: Evaluation of activity of potencional antibiotic substances through the use of microdilution broth method II Background: To evaluate the antibacterial activity of the compounds synthesized by the Department of Pharmaceutical Chemistry and Drug control, Faculty of Pharmacy in Hradec Králové, Charles University in Prague under the leadership of prof. PharmDr. Martin Doležal, Ph.D. Methods: Using broth microdilution method was tested antibacterial activity at eight bacterial strains, namely Staphylococcus aureus, Staphylococcus aureus methicilin resistant, Staphylococcus epidermidis, Enterococcus sp., Escherichia coli, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL positive and Pseudomonas aeruginosa. Results: Test substances were divided into 5 groups according to similarities in the chemical structure. The best antibacterial activity was observed in the group of derivatives of N-benzyl- 6-(alkylamino)pyrazine-2-carboxamide. Conclusion: Antibacterial activity was detected in 10 of 24 tested compounds. From active substances, all but one danced only Gram-positive bacteria. Among...

Under vissa omständigheter kan bakterier kan ta sig in i blodbanan där de kan orsaka allvarliga infektioner (bakteriemi). Metoden som används för identifiering och resistensbestämning av bakterier i blododlingar kräver minst 16 timmars inkubation. Fram tills en resistensbestämning utförts kan empirisk antibiotikabehandling användas, men med ökande resistensutbredning blir det alltmer osäkert om denna behandling är verksam. Nyligen har en lappdiffusionsmetod för resistensbestämning direkt från blododling validerats, som kan läsas av efter 4, 6 och/eller 8 timmars inkubation. Det finns även publicerade arbeten där flödescytometriassisterad resistensbestämning används, men då krävs att bakterierna finns i tillräckligt hög koncentration, befinner sig i tillväxtfas och finns som renkultur. Syftet med examensarbetet var att optimera hantering av blododlingsflaskor så att bakterier från blododlingsflaskorna kunde isoleras med tillräcklig kvalitet och koncentration för att kunna utföra resistensbestämning med flödescytometri. Upprening av bakterierna utfördes med olika tvättbuffertar och sedan utfördes resistensbestämning med flödescytometri och  referensmetoden buljongspädning. Resultaten från uppreningen visade att sterilt vatten och Tween20 gynnade bakteriernas återhämtningsförmåga mest. Resistensbestämning utfördes med Klebsiella pneumoniae ATCC700603,  K. pneumoniae CCUG56233 och K. pneumoniae ATCC13882, som tvättats med sterilt vatten och Tween20. För CCUG56233 skiljde 1 spädningssteg i koncentrationsskalan mellan metoderna. ATCC-isolaten erhöll likartade MIC-värden (minimum inhibitory concentration) vid alla analyser men där fanns en skillnad på 2 spädningssteg mellan buljongspädning och analys med flödescytometri. Detta kan förklaras av skillnaden i inkubationstid mellan metoderna. Slutsatsen som kan dras är därför att resultaten från de två metoderna vid resistensbestämning är likartade och att sterilt vatten är mest lämpligt att använda vid upprening av bakterier. Fler undersökningar bör dock utföras.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Introdução: Pneumonias causadas por Staphylococcus aureus meticilina resistentes (Methicillin resistant Staphylococcus aureus - MRSA) estão associadas com uma alta mortalidade em pacientes internados em Centros de Terapia Intensiva (CTI). Não é incomum que ocorra falha no tratamento de pneumonia por MRSA com vancomicina. Alguns estudos têm mostrado que há uma relação entre mortalidade em infecções por MRSA e concentrações inibitórias mínimas (CIM) elevadas para vancomicina por Etest®, apesar dos isolados serem sensíveis à vancomicina (CIM ≤2μg/mL), conforme o Clinical Laboratory Standards Institute (CLSI). Objetivos: O objetivo deste estudo foi avaliar as CIMs para vancomicina em pacientes com diagnóstico de pneumonia por MRSA, descrever a relação entre a CIM da vancomicina obtido por Etest® e microdiluição em caldo com mortalidade em 30 dias em pacientes com pneumonia por MRSA internados em um hospital terciário, acadêmico, no sul do Brasil. Métodos: Estudo prospectivo de coorte. Foram incluídos todos os pacientes com pneumonia adquirida no hospital (hospital adquired pneumonia - HAP) e pneumonia associada à ventilação (ventilator associated pneumonia - VAP) por MRSA entre Junho de 2009 e Dezembro de 2011. A CIM da vancomicina do primeiro isolado do trato respiratório foi determinado por Etest® e microdiluição em caldo. As variáveis selecionadas para serem analisadas incluíram idade, sexo, comorbidades, dosagem sérica de vancomicina, escore APACHE II (Acute Physiological and Chronic Health Evaluation II) e a presença do accessory gene regulator II (agr II). O desfecho principal foi morte em 30 dias. Resultados: Foram incluídos 85 pacientes. A mortalidade em 30 dias foi de 42,4%. Em sessenta e oito pacientes (80,1%) as CIMs da vancomicina obtidos por Etest® foram menores ou iguais a 1,0μg/mL, enquanto todas as CIMs (N=85) obtidas por microdiluição em caldo foram ≤1,0μg/mL. Não houve correlação entre as CIMs por Etest® e microdiluição em caldo com morte em 30 dias. Quarenta e seis isolados de MRSA (54,1%) foram positivos para o locus agr II. A média da vancomicina sérica foi 20,7μg/mL (12,0 – 29,0) e a média do escore APACHE II foi 23,0 (17,5 – 29,0). O escore APACHE II foi relacionado com mortalidade em 30 dias. Conclusões: As CIMs da vancomicina obtidas por Etest® e microdiluição em caldo não foram associadas com altos níveis de mortalidade em pacientes com pneumonia grave por MRSA. / Background: Pneumonias due to methicillin resistant Staphylococcus aureus (MRSA) are associated with significant mortality in Intensive Care Units (ICU) patients. Vancomycin treatment failure in MRSA pneumonia patients is not uncommon. Some reports found a link between high mortality in MRSA infections and higher vancomycin minimum inhibitory concentrations (MICs) by Etest®, despite the susceptibility of the isolates (MIC ≤2,0μg/mL), according to the Clinical Laboratory Standards Institute (CLSI). Objectives: The purpose of this study was evaluate vancomycin MICs in patients with MRSA pneumonia, describe the relationship between vancomycin MIC generated by Etest® and broth microdilution with 30-day mortality in MRSA pneumonia patients. Methods: We conducted a prospective cohort study. All patients with MRSA hospital acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) admitted in ICU between June 2009 and December 2011 were included. Vancomycin MIC for the first isolate of the respiratory tract was determined by Etest® and broth microdilution. The variables selected for the analysis included age, sex, comorbid conditions, vancomycin serum trough concentration, APACHE II (Acute Physiological and Chronic Health Evaluation II) score and the presence of the accessory gene regulator II (agr II). The primary outcome was mortality at 30-day. Results: Eithy five patients were included. All case mortality at the 30-day was 42.4%. Sixty eight of the patients (80.1%) of the vancomycin MICs generated by Etest® were equal or below 1.0μg/mL. While all MICs generated by broth microdilution were ≤1.0μg/mL. MICs generated by Etest® and broth microdilution were not associated with 30-day mortality. Forty six isolates of MRSA (54.1%) from patients were positive for agr II. The median serum vancomycin was 20.7μg/mL (12.0 – 29.0) and the median APACHE II score was 23.0 (17.5 – 29.0). The APACHE II score was related to 30-day mortality. Conclusions: Vancomycin MICs by Etest® and broth microdilution were not associated with higher mortality leves in patients with severe MRSA pneumonia.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Specialist in Laboratory Methods Candidate: Bc. Lucie Davidová Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: In vitro screening of novel potentially active antibacterial compounds. The diploma thesis deals with the antibiotics policy and is focused on resistance and mechanism of action of the antibiotics. A great attention is also paid to novel substances testing in terms of their impact on the selected bacteria. Theoretical part of thesis describes the system of antibiotic policy including its structure and organization in the Czech Republic. It also briefly summarizes this issue globally as well as individual organizations involved in the organization of the antibiotic policy. The theoretical part is also concerned with the history, classification, effects and significance of the antibiotics. A particular attention is paid to the chapter describing the macromolecular mechanism of action of 2 selected groups of substances (antibiotics) on bacteria, including physiological functions of some bacterial structures. And, last but not least, the theoretical part summarizes principles of bacterial resistance including its origin and development. The diploma thesis...

A própolis é um material resinoso coletado pelas abelhas de diversas partes das plantas como brotos, folhas jovens e exsudatos. É utilizado para embalsamar animais que tenham morrido dentro da colmeia, vedar as paredes e conferir proteção frente a doenças. O emprego desse produto natural com finalidade medicinal remonta a antiguidade, tendo iniciado junto com antigas civilizações como a Egípcia, Grega e Romana e perduram até os dias atuais com ampla utilização em múltiplas finalidades terapêuticas médicas e veterinárias. O objetivo desse trabalho foi avaliar o potencial antifúngico, a capacidade de remover e inibir biofilmes de espécies de Candida não-albicans de diferentes tipos de própolis e analisar a composição química da própolis obtida do Rio Grande do Sul (RS). Foram realizadas macerações e extrações sequenciais com solventes em ordem crescente de polaridade a fim de purificar os extratos. Em seguida foi feita a determinação da atividade antifúngica pelo método da microdiluição em caldo e o ensaio de remoção e inibição de biofilmes através de teste em corpo de prova (utilizando cateter como material teste). Após, foi realizada a análise química da fração diclorometano de própolis obtida do RS. De modo geral, os extratos e frações de própolis vermelha e própolis nativa do RS demonstraram forte atividade anti-Candida frente às leveduras testadas e significativa capacidade de remoção de seu biofilme aderido em cateteres, como corpo de prova. A própolis verde apresentou baixo espectro de ação antifúngica. A análise química da fração diclorometano de própolis obtida do RS levou ao isolamento dos flavonoides pinocembrina, pinostrobina e pinostrobina chalcona. Os resultados evidenciam o potencial da própolis nativa do RS e da própolis vermelha para o desenvolvimento de novos agentes antifúngicos e desinfetantes contra Candida não-albicans. / Propolis is a resinous material collected by bees from various plant parts such as buds, young leaves and exudates. It is used to embalm animals that have died inside the hive, seal the walls and confer protection against the disease. The use of this natural product with medicinal purposes dates back to antiquity, having started with ancient civilizations as the Egyptian, Greek and Roman, and lasted to the present day with extensive use in multiple medical and veterinary therapeutic purposes. The aim of this study was to evaluate the antifungal potential, the ability to remove and inhibit biofilms of Candida non-albicans of different types of propolis and analyze the chemical composition of propolis obtained from Rio Grande do Sul (RS). Maceration and sequential extractions with solvents in order of increasing polarity were performed to purify the extracts. Then the antifungal activity was determined by the method of broth microdilution testing and removal and inhibition of biofilms through test of body of proof (catheter using as test material). After, the chemical analysis of the dichloromethane fraction of propolis obtained in RS was performed. In general, the extracts and fractions of propolis of RS demonstrated strong anti-Candida activity across the tested yeasts and significant capacity removal of biofilm attached in catheter, as test body. The green propolis had low action spectrum antifungal. Chemical analysis of the dichloromethane fraction of propolis native to RS led to the isolation of the flavonoids pinocembrin, chalcone pinostrobin and pinostrobin. The results show the potential of the RS native propolis and red propolis for the development of new antifungal agents and disinfectants against non-albicans Candida.